PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions

Choice of methodology and surrogate prey are decisive for the quality of protistan bacterivory rate estimates

Microeukaryote predation on bacteria is a fundamental phenomenon to understand energy and nutrient dynamics at the base of the aquatic food web. To date, the most prevalent way to estimate grazing rates is by using epifluorescence microscopy to enumerate ingestion events of fluorescently labelled tracers (FLTs) after short-term incubation experiments. However, this approach can be sensitive to the type of FLT, requires skillful preparation of the samples and is limited to small sample sizes. We tested the susceptibility of rate estimates to the choice of prey and made a side-by-side comparison between microscopy and flow cytometry when recording ingestion by a bacterivorous flagellate. Short-term uptake experiments were established using 5 types of FLTs differing in quality (living, dead or inert) and size (large or small), with Ochromonas triangulata as a model flagellate. The experiments showed that (1) each of the different prey types yielded different clearing rates, ranging from 0.5 to 3.6 nl cell-1 h-1, with the largest differences (3-fold or higher) between small prey (lower rates) and large prey (higher rates); (2) the cytometry estimate differed significantly from the microscopy estimate in 3 out of 4 experimental configurations; and (3) the precision of the cytometric analysis was greater, with >3-fold higher uncertainty associated with microscopy counting. Our results validate that flow cytometry provides a more precise bacterivory estimate, and that the choice of FLT influences the grazing rate estimate to a high extent regardless of the analytical method used

Goldberger-Treiman relation and Wu-type experiment in the decuplet sector

The leading-order chiral Lagrangian for the baryon octet and decuplet states coupled to Goldstone bosons and external sources contains six low-energy constants. Five of them are fairly well known from phenomenology, but the sixth one is practically unknown. This coupling constant provides the strength of the (p-wave) coupling of Goldstone bosons to decuplet states. Its size and even sign are under debate. Quark model and QCD for a large number of colors provide predictions, but some recent phenomenological analyses suggest even an opposite sign for the Delta-pion coupling. The Goldberger-Treiman relation connects this coupling constant to the axial charge of the Delta baryon. This suggests a Wu-type experiment to determine the unknown low-energy constant. While this is not feasible in the Delta sector because of the large hadronic width of the Delta, there is a flavor symmetry related process that is accessible: the weak semileptonic decay of the Omega baryon to a spin 3/2 cascade baryon. A broad research program is suggested that can pin down at least the rough size and the sign of the last unknown low-energy constant of the leading-order Lagrangian. It encompasses experimental measurements, in particular the forward-backward asymmetry of the semileptonic decay, together with a determination of the quark-mass dependences using lattice QCD for the narrow decuplet states and chiral perturbation theory to extrapolate to the Delta sector. Besides discussing the strategy of the research program, the present work provides a feasibility check based on a simple leading-order calculation.Comment: 7 page

mOTUpan: a robust Bayesian approach to leverage metagenome-assembled genomes for core-genome estimation

Recent advances in sequencing and bioinformatics have expanded the tree of life by providing genomes for uncultured environmentally relevant clades, either through metagenome-assembled genomes or through single-cell genomes. While this expanded diversity can provide novel insights into microbial population structure, most tools available for core-genome estimation are sensitive to genome completeness. Consequently, a major portion of the huge phylogenetic diversity uncovered by environmental genomic approaches remains excluded from such analyses. We present mOTUpan, a novel iterative Bayesian method for computing the core genome for sets of genomes of highly diverse completeness range. The likelihood for each gene cluster to belong to core or accessory genome is estimated by computing the probability of its presence/absence pattern in the target genome set. The core-genome prediction is computationally efficient and can be scaled up to thousands of genomes. It has shown comparable estimates to state-of-the-art tools Roary and PPanGGOLiN for high-quality genomes and is capable of using genomes at lower completeness thresholds. mOTUpan wraps a bootstrapping procedure to estimate the quality of a specific core-genome prediction, as the accuracy of each run will depend on the specific completeness distribution and the number of genomes in the dataset under scrutiny. mOTUpan is implemented in the mOTUlizer software package, and available at github.com/moritzbuck/mOTUlizer, under GPL 3.0 license

Exploring environmental intra-species diversity through non-redundant pangenome assemblies

At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different environmental niches and can have a profound influence on microbial community dynamics. As a consequence, any individual genome or population will contain merely a fraction of the total genetic diversity of any operationally defined "species", whose ecological potential can thus be only fully understood by studying all of their genomes and the genes therein. This concept, known as the pangenome, is valuable for studying microbial ecology and evolution, as it partitions genomes into core (present in all the genomes from a species, and responsible for housekeeping and species-level niche adaptation among others) and accessory regions (present only in some, and responsible for intra-species differentiation). Here we present SuperPang, an algorithm producing pangenome assemblies from a set of input genomes of varying quality, including metagenome-assembled genomes (MAGs). SuperPang runs in linear time and its results are complete, non-redundant, preserve gene ordering and contain both coding and non-coding regions. Our approach provides a modular view of the pangenome, identifying operons and genomic islands, and allowing to track their prevalence in different populations. We illustrate this by analysing intra-species diversity in Polynucleobacter, a bacterial genus ubiquitous in freshwater ecosystems, characterized by their streamlined genomes and their ecological versatility. We show how SuperPang facilitates the simultaneous analysis of allelic and gene content variation under different environmental pressures, allowing us to study the drivers of microbial diversification at unprecedented resolution

Streamlined and Abundant Bacterioplankton Thrive in Functional Cohorts

While fastidious microbes can be abundant and ubiquitous in their natural communities, many fail to grow axenically in laboratories due to auxotrophies or other dependencies. To overcome auxotrophies, these microbes rely on their surrounding cohort. A cohort may consist of kin (ecotypes) or more distantly related organisms (community) with the cooperation being reciprocal or nonreciprocal and expensive (Black Queen hypothesis) or costless (by-product). These metabolic partnerships (whether at single species population or community level) enable dominance by and coexistence of these lineages in nature. Here we examine the relevance of these cooperation models to explain the abundance and ubiquity of the dominant fastidious bacterioplankton of a dimictic mesotrophic freshwater lake. Using both culture-dependent (dilution mixed cultures) and culture-independent (small subunit [SSU] rRNA gene time series and environmental metagenomics) methods, we independently identified the primary cohorts of actinobacterial genera "Candidatus Planktophila" (acI-A) and "Candidatus Nanopelagicus" (acI-B) and the proteobacterial genus "Candidatus Fonsibacter" (LD12). While "Ca. Planktophila" and "Ca. Fonsibacter" had no correlation in their natural habitat, they have the potential to be complementary in laboratory settings. We also investigated the bifunctional catalase-peroxidase enzyme KatG (a common good which "Ca. Planktophila" is dependent upon) and its most likely providers in the lake. Further, we found that while ecotype and community cooperation combined may explain "Ca. Planktophila" population abundance, the success of "Ca. Nanopelagicus" and "Ca. Fonsibacter" is better explained as a community by-product. Ecotype differentiation of "Ca. Fonsibacter" as a means of escaping predation was supported but not for overcoming auxotrophies.IMPORTANCE This study examines evolutionary and ecological relationships of three of the most ubiquitous and abundant freshwater bacterial genera: "Ca. Planktophila" (acI-A), "Ca. Nanopelagicus" (acI-B), and "Ca. Fonsibacter" (LD12). Due to high abundance, these genera might have a significant influence on nutrient cycling in freshwaters worldwide, and this study adds a layer of understanding to how seemingly competing clades of bacteria can coexist by having different cooperation strategies. Our synthesis ties together network and ecological theory with empirical evidence and lays out a framework for how the functioning of populations within complex microbial communities can be studied

Thiobacillus as a key player for biofilm formation in oligotrophic groundwaters of the Fennoscandian Shield

Biofilm formation is a common adaptation for microbes in energy-limited conditions such as those prevalent in the vast deep terrestrial biosphere. However, due to the low biomass and the inaccessible nature of subsurface groundwaters, the microbial populations and genes involved in its formation are understudied. Here, a flow-cell system was designed to investigate biofilm formation under in situ conditions in two groundwaters of contrasting age and geochemistry at the aspo Hard Rock Laboratory, Sweden. Metatranscriptomes showed Thiobacillus, Sideroxydans, and Desulforegula to be abundant and together accounted for 31% of the transcripts in the biofilm communities. Differential expression analysis highlighted Thiobacillus to have a principal role in biofilm formation in these oligotrophic groundwaters by being involved in relevant processes such as the formation of extracellular matrix, quorum sensing, and cell motility. The findings revealed an active biofilm community with sulfur cycling as a prominent mode of energy conservation in the deep biosphere

Large-scale phylogenomics of aquatic bacteria reveal molecular mechanisms for adaptation to salinity

The crossing of environmental barriers poses major adaptive challenges. Rareness of freshwater-marine transi-tions separates the bacterial communities, but how these are related to brackish counterparts remains elusive, as do the molecular adaptations facilitating cross-biome transitions. We conducted large-scale phylogenomic analysis of freshwater, brackish, and marine quality-filtered metagenome-assembled genomes (11,248). Average nucleotide identity analyses showed that bacterial species rarely existed in multiple biomes. In contrast, distinct brackish basins cohosted numerous species, but their intraspecific population structures displayed clear signs of geographic separation. We further identified the most recent cross-biome transitions, which were rare, ancient, and most commonly directed toward the brackish biome. Transitions were accompanied by systematic changes in amino acid composition and isoelectric point distributions of inferred proteomes, which evolved over millions of years, as well as convergent gains or losses of specific gene functions. Therefore, adaptive chal-lenges entailing proteome reorganization and specific changes in gene content constrains the cross-biome tran-sitions, resulting in species-level separation between aquatic biomes

Homogenisation of water and sediment bacterial communities in a shallow lake (lake Balihe, China)

Planktonic and benthic bacterial communities hold central roles in the functioning of freshwater ecosystems and mediate key ecosystem services such as primary production and nutrient remineralisation. Although it is clear that such communities vary in composition both within and between lakes, the environmental factors and processes shaping the diversity and composition of freshwater bacteria are still not fully understood. In order to assess seasonal and spatial variability in lake bacterial communities and identify environmental factors underpinning biogeographical patterns, we performed a large-scale sampling campaign with paired water and sediment sample collection at 18 locations during four seasons in Lake Balihe, a subtropical shallow fish-farming lake in mid-eastern China. Pelagic and benthic bacterial communities were distinctly different in terms of diversity, taxonomic composition and community structure, with Actinobacteria, Bacteroidetes, Cyanobacteria and Alphaproteobacteria dominating lake water, and Acidobacteria, Bacteroidetes, Chloroflexi, Gammaproteobacteria and Deltaproteobacteria dominating sediment. Nevertheless, these two communities had stronger spatial concordance and overlap in taxa during spring and autumn seasons. Together, the main drivers of both the spatial and temporal variations in Lake Balihe bacterial communities were identified as water temperature, turbidity, nitrogen and phosphorus availability, and thermal stratification controlled by wind-mixing and activity of the dense farmed fish populations. Notably, populations affiliated with Firmicutes, known to be abundant in fish gut microbiome, were especially abundant in the summer season and locations where high fish biomass was found, suggesting a potential link between fish gut microbiome and the pelagic bacterial communities. Our findings demonstrated seasonal homogenisation of pelagic and benthic bacterial communities linked to marked shifts in a set of seasonally-driven environmental variables including water temperature and nutrient availability